This invention is directed to a process for inactivating viruses in blood or blood products, comprising: adding a phenothiazine dye to the solutions or suspensions to be treated and subsequently irradiating said phenothiazine dye-containing solutions or suspensions with visible light in the range of the adsorption peak of the respective dye whereafter the blood or blood products may be passed over an adsorbing agent for removal of the dye.
FIG. 1 of the accompanying drawings is a graph of time v. inactivation of human immunodeficiency virus (HIV-1) by the method of this invention.
FIG. 2 is a graph illustrating the influence of light intensity on the inactivation of vesicular stomatitis virus (VSV) by the method of this invention.
FIG. 3 is a bar graph showing the efficacy of various pheno-thiazine dyes for photodynamic inactivation of VSV in platelet suspensions at dye concentrations of 1 xcexcM in each specimen and an irradiation time of 30 minutes.
FIG. 4 is a graph showing the rate of adsorption of methylene blue from a 5% albumin solution by various adsorbents.
It is known that photodynamic substances in combination with visible or ultraviolet light may have a virus inactivating effect. This is due to the affinity of these substances to external virus structures or to the viral nucleic acid. Heinmets, F. et al, Inactivation of Viruses in Plasma by Photosensitized Oxidation, Walter Reed Research Report 53-55 (1955) showed that Eastern equine encephalomyelitis (EEE) virus in plasma was inactivated by various phenothiazine dyes at a dye concentration of 10 xcexcM on activation with visible light. Toluidine blue 0 at a concentration of 2.5 xcexcM effectively inactivated EEE virus in plasma in 1 minute using a 1000 watt light source in a special apparatus, whereas lesser dye concentrations failed to inactivate the virus completely.
Phenothiazine dyes react with membrane structures of enveloped viruses and damage the same irreversibly under the action of light, whereby the virus looses its infectiousness (cf. Snipes, W. et al, 1979, Photochem. and Photobiol. 29, 785-790). However, photodynamic substances also interact with viral RNA or DNA, especially with the guanine residues. When a dye/nucleic acid-complex has been formed, it is stimulated by light energy so that denaturation of the nucleic acid and finally strand breakages result. The reason is that phenothiazine dyes cause the conversion of molecular oxygen to activated species (singlet oxygen, oxygen radicals, etc.) which are highly reactive and may have various virucidal effects (cf. Hiatt, C. W., 1972, in: Concepts in Radiation Cell Biology, pp. 57-89, Academic Press, New York; Oh Uigin et al, 1987, Nucl. Acid. Res. 15, 7411-7427).
In contrast to other photodynamic dyes for virus inactivation, phenothiazine dyes, particularly methylene blue (MB), neutral red, thionine, and toluidine blue (TB) are of special interest because they may, in combination with visible light, inactivate a number of viruses, including some viruses which do not possess a lipid envelope, e.g. adenovirus. Other photoactive dyes, e.g. azure A, azure B, azure C, merocyanin 540, hematoporphyrin IX dihydrochloride, aluminum phthalocyanine chloride, acridine orange, and psoralens may also be useful.
In addition to that, MB and TB for instance, have themselves been used therapeutically, among other uses also as antidotes to carbon-monoxide poisoning and in long-term therapy of psychotic diseases. In this connection, quantities of MB or TB much higher than those required for virus inactivation are used (1 to 2 mg/kg of body weight) without any significant side effects. The low toxicities of MB and TB are also substantiated by data obtained from animal experiments.
However, since 1955, those skilled in the art have assumed that dye concentrations especially in the case of TB, of less than 2.5 xcexcM, have only an insufficient virus inactivating effect (cf. F. Heinmets et al, 1955, Joint Report with the Naval Medical Research Institute, Walter Reed Army Institute of Research, U.S.A.).
In the previously described investigations of virus inactivation with phenothiazine dyes, the dye concentrations are between 10 xcexcM and 100 xcexcM (Chang and Weinstein, 1975, Photodynamic Inactivation of Herpes virus Hominis by Methylene Blue. Proceedings of the Society for Experimental Biology and Medicine 148, 291-293; Yen and Simon, 1978, Photosensitization of Herpes Simplex Virus Type 1 with Neutral Red, J. gen. Virol., 41, 273-271). At these concentrations, there arises the drawback that not only viruses may be inactivated, but also plasma proteins such as the coagulation factors, may be damaged. This is one of the reasons why phenothiazine dyes have so far not achieved any significance for virus inactivation in blood or blood products.
It is an object of the subject invention to provide a method in which various kinds of viruses are killed by the use of selected phenothiazine dyes without substantial functionally detrimental effects on the plasma proteins. It is a further object of the subject invention that the said process be of simple design, such that blood or blood products may be subjected to direct treatment in commercially available blood bags. At the low level concentrations of dye used in such treatment, removal of the dye is not necessary. However, a method is provided so that the added dyes may be removed after treatment if so desired.
The specified objects are accomplished in accordance with the invention in that these dyes are used at a concentration of from about 0.1 to about 2 xcexcM, preferably from about 0.5 to 2 xcexcM, and irradiation is effected directly in transparent containers, for example, blood bags of the kind used for collection and storage of blood or blood products.
Irradiation is performed either with daylight of sufficient intensity or an electric light, preferably from a cold light source, at a wavelength in the range of the absorption peak of the respective dye. Also, the following conditions should be observed for virus inactivation in blood plasma or plasma protein solutions. The operating temperature should range from 0xc2x0 to 37xc2x0 C., preferably from 4xc2x0 to 20xc2x0 C. The inactivating time may range from 2 minutes to 5 hours, preferably from 5 minutes to 3 hours, and pH should be between 5 and 9, preferably between pH 6 and pH 8.
An outstanding advantage of the process of this invention lies in its simplicity. F. Heinmets et al (as specified above) describe a highly complex apparatus through which, for instance, blood plasma must be passed. Here, problems of maintenance and above all capacity problems occur. Surprisingly, it has now been found that substantially smaller amounts of dye than that used in the prior art are sufficient for substantially complete inactivation of many viruses and that no complex technical apparatus is required for photoinactivation of viruses in blood or blood products.
Unexpectedly, it has also been found that non-enveloped viruses, such as adenovirus, which are normally resistant to photodynamic treatment, could be made photo-sensitive by a freezing/thawing step and subsequently could be inactivated in accordance with this invention. Inactivation has been successfully carried out irrespective of the order of freezing/thawing steps and addition of the dye. Freezing here means a deep-freezing operation at temperatures from about xe2x88x9220xc2x0 C. to about 80xc2x0 K. Normally, deep-freezing is carried out at or below xe2x88x9230xc2x0 C.
Virus inactivation may be carried out directly in blood or plasma bags although these are transparent only to a limited extent. It is merely necessary to add the dye. Then the bag inclusive of its contents is exposed to light, whereafter the respective product may be used or further processed.
In one specific embodiment of the invention, the process is carried out in two interconnected containers, preferably containers suitable for collecting and storing blood or plasma. A separating column containing an agent for selectively absorbing the phenothiazine dye is interposed in the connecting passageway between the containers so that the dye is removed from the blood or plasma as it passes from the first container to the second container.
Thus, the process can be carried out without any major technical effort and is excellently suited for integration in the processing of individual blood donations. The small quantity of the dye used may either remain in the treated fluid or may be removed by adsorbing agents.
Suitable blood or blood products include:
whole blood
red cell concentrates
platelet concentrates
plasma
serum
cryoprecipitate
concentrates of coagulation factors
inhibitors
cold insoluble globulin
albumin
fibrinogen
immune globulin.